Methods for the identification of inhibitors of threonine synthase as antibiotics

ABSTRACT

The present inventors have discovered that Threonine synthase is essential for fungal pathogenicity. Specifically, the inhibition of Threonine synthase gene expression in fungi results in no signs of successful infection or lesions. Thus, Threonine synthase can be used as a target for the identification of antibiotics, preferably antifungals. Accordingly, the present invention provides methods for the identification of compounds that inhibit Threonine synthase expression or activity. The methods of the invention are useful for the identification of antibiotics, preferably antifungals.

FIELD OF THE INVENTION

[0001] The invention relates generally to methods for the identificationof antibiotics, preferably antifungals that affect the biosynthesis ofL-threonine.

BACKGROUND OF THE INVENTION

[0002] Filamentous fungi are the causal agents responsible for manyserious pathogenic infections of plants and animals. Since fungi areeukaryotes, and thus more similar to their host organisms than, forexample bacteria, the treatment of infections by fungi poses specialrisks and challenges not encountered with other types of infections. Onesuch fungus is Magnaporthe grisea, the fungus that causes rice blastdisease. It is an organism that poses a significant threat to foodsupplies worldwide. Other examples of plant pathogens of economicimportance include the pathogens in the genera Agaricus, Alternaria,Anisogramma, Anthracoidea, Antrodia, Apiognomonia, Apiosporina,Armillaria, Ascochyta, Aspergillus, Bipolaris, Bjerkandera,Botryosphaeria, Botrytis, Ceratobasidium, Ceratocystis, Cercospora,Cercosporidium, Cerotelium, Cerrena, Chondrostereum, Chryphonectria,Chrysomyxa, Cladosporium, Claviceps, Cochliobolus, Coleosporium,Colletotrichium, Colletotrichum, Corticium, Corynespora, Cronartium,Cryphonectria, Cryptosphaeria, Cyathus, Cymadothea, Cytospora,Daedaleopsis, Diaporthe, Didymella, Diplocarpon, Diplodia,Discohainesia, Discula, Dothistroma, Drechslera, Echinodontium, Elsinoe,Endocronartium, Endothia, Entyloma, Epichloe, Erysiphe, Exobasidium,Exserohilum, Fomes, Fomitopsis, Fusarium, Gaeumannomyces, Ganoderma,Gibberella, Gloeocercospora, Gloeophyllum, Gloeoporus, Glomerella,Gnomoniella, Guignardia, Gymnosporangium, Helminthosporium,Herpotrichia, Heterobasidion, Hirschioporus, Hypodermella, Inonotus,Irpex, Kabatiella, Kabatina, Laetiporus, Laetisaria, Lasiodiplodia,Laxitextum, Leptographium, Leptosphaeria, Leptosphaerulina,Leucytospora, Linospora, Lophodermella, Lophodermium, Macrophomina,Magnaporthe, Marssonina, Melampsora, Melampsorella, Meria, Microdochium,Microsphaera, Monilinia, Monochaetia, Morchella, Mycosphaerella,Myrothecium, Nectria, Nigrospora, Ophiosphaerella, Ophiostoma,Penicillium, Perenniporia, Peridermium, Pestalotia, Phaeocryptopus,Phaeolus, Phakopsora, Phellinus, Phialophora, Phoma, Phomopsis,Phragmidium, Phyllachora, Phyllactinia, Phyllosticta, Phymatotrichopsis,Pleospora, Podosphaera, Pseudopeziza, Pseudoseptoria, Puccinia,Pucciniastrum, Pyricularia, Rhabdocline, Rhizoctonia, Rhizopus,Rhizosphaera, Rhynchosporium, Rhytisma, Schizophyllum, Schizopora,Scirrhia, Sclerotinia, Sclerotium, Scytinostroma, Septoria, Setosphaera,Sirococcus, Spaerotheca, Sphaeropsis, Sphaerotheca, Sporisorium,Stagonospora, Stemphylium, Stenocarpella, Stereum, Taphrina,Thielaviopsis, Tilletia, Trametes, Tranzschelia, Trichoderma, Tubakia,Typhula, Uncinula, Urocystis, Uromyces, Ustilago, Valsa, Venturia,Verticillium, Xylaria, and others. Related organisms in theclassification, oomycetes, that include the genera Albugo, Aphanomyces,Bremia, Peronospora, Phytophthora, Plasmodiophora, Plasmopara,Pseudoperonospora, Pythium, Sclerophthora, and others are alsosignificant plant pathogens and are sometimes classified along with thetrue fungi. Human diseases that are caused by filamentous fungi includelife-threatening lung and disseminated diseases, often a result ofinfections by Aspergillus fumigatus. Other fungal diseases in animalsare caused by fungi in the genera, Fusarium, Blastomyces, Microsporum,Trichophyton, Epidermophyton, Candida, Histoplamsa, Pneumocystis,Cryptococcus, other Aspergilli, and others. The control of fungaldiseases in plants and animals is usually mediated by chemicals thatinhibit the growth, proliferation, and/or pathogenicity of the fungalorganisms. To date, there are less than twenty known modes-of-action forplant protection fungicides and human antifungal compounds.

[0003] A pathogenic organism has been defined as an organism thatcauses, or is capable of causing disease. Pathogenic organisms propagateon or in tissues and may obtain nutrients and other essential materialsfrom their hosts. A substantial amount of work concerning filamentousfungal pathogens has been performed with the human pathogen, Aspergillusfumigatus. Shibuya et al. (Shibuya, K., M. Takaoka, et al. (1999) MicrobPathog 27: 123-31 (PMID: 10455003)) have shown that the deletion ofeither of two suspected pathogenicity related genes encoding an alkalineprotease or a hydrophobin (rodlet) respectively, did not reducemortality of mice infected with these mutant strains. Smith et al.(Smith, J. M., C. M. Tang, et al. (1994) Infect Immun 62: 5247-54 (PMID:7960101)) showed similar results with alkaline protease and theribotoxin restrictocin; Aspergillus fumigatus strains mutated for eitherof these genes were fully pathogenic to mice. Reichard et al. (Reichard,U., M. Monod, et al. (1997) J Med Vet Mycol 35: 189-96 (PMID: 9229335))showed that deletion of the suspected pathogenicity gene encodingaspergillopepsin (PEP) in Aspergillus fumigatus had no effect onmortality in a guinea pig model system, and Aufauvre-Brown et al(Aufauvre-Brown, A., E. Mellado, et al. (1997) Fungal Genet Biol 21:141-52 (PMID: 9073488)) showed no effects of a chitin synthase mutationon pathogenicity. However, not all experiments produced negativeresults. Ergosterol is an important membrane component found in fungalorganisms. Pathogenic fungi that lack key enzymes in this biochemicalpathway might be expected to be non-pathogenic since neither the plantnor animal hosts contain this particular sterol. Many antifungalcompounds that affect this biochemical pathway have been described(Onishi, J. C. and A. A. Patchett (1990a, b, c, d, and e) U.S. Pat. Nos.4,920,109; 4,920,111; 4,920,112; 4,920,113; and 4,921,844, Merck & Co.Inc. (Rahway N.J.)) and (Hewitt, H. G. (1998) Fungicides in CropProtection Cambridge, University Press). D'Enfert et al. (D'Enfert, C.,M. Diaquin, et al. (1996) Infect Immun 64: 4401-5 (PMID: 8926121))showed that an Aspergillus fumigatus strain mutated in an orotidine5′-phosphate decarboxylase gene was entirely non-pathogenic in mice, andBrown et al. (Brown, J. S., A. Aufauvre-Brown, et al. (2000) MolMicrobiol 36:1371-80 (PMID: 10931287)) observed a non-pathogenic resultwhen genes involved in the synthesis of para-aminobenzoic acid weremutated. Some specific target genes have been described as havingutility for the screening of inhibitors of plant pathogenic fungi. Bacotet al. (Bacot, K. O., D. B. Jordan, et al. (2000) U.S. Pat. No.6,074,830, E. I. du Pont de Nemours & Company (Wilmington Del.))describe the use of 3,4-dihydroxy-2-butanone 4-phosphate synthase, andDavis et al. (Davis, G. E., G. D. Gustafson, et al. (1999) U.S. Pat. No.5,976,848, Dow AgroSciences LLC (Indianapolis Ind.)) describe the use ofdihydroorotate dehydrogenase for potential screening purposes.

[0004] There are also a number of papers that report less clear results,showing neither full pathogenicity nor non-pathogenicity of mutants.Hensel et al. (Hensel, M., H. N. Arst, Jr., et al. (1998) Mol Gen Genet258: 553-7 (PMID: 9669338)) showed only moderate effects of the deletionof the area transcriptional activator on the pathogenicity ofAspergillus fumigatus.

[0005] Therefore, it is not currently possible to determine whichspecific growth materials may be readily obtained by a pathogen from itshost, and which materials may not. We have found that Magnaporthe griseathat cannot synthesize their own L-threonine are non-pathogenic on theirhost organism. To date there do not appear to be any publicationsdemonstrating an anti-pathogenic effect of the knock-out,overexpression, antisense expression, or inhibition of the genes or geneproducts involved in L-threonine biosynthesis in filamentous fungi.Thus, it has not been shown that the de novo biosynthesis of L-threonineis essential for fungal pathogenicity. Thus, it would be desirable todetermine the utility of the enzymes involved in L-threoninebiosynthesis for evaluating antibiotic compounds, especially fungicides.If a fungal biochemical pathway or specific gene product in that pathwayis shown to be required for fungal pathogenicity, various formats of invitro and in vivo screening assays may be put in place to discoverclasses of chemical compounds that react with the validated target gene,gene product, or biochemical pathway, and are thus candidates forantifungal, biocide, and biostatic materials.

SUMMARY OF THE INVENTION

[0006] Surprisingly, the present inventors have discovered that in vivodisruption of the gene encoding Threonine synthase in Magnaporthe griseaprevents or inhibits the pathogenicity of the fungus. Thus, the presentinventors have discovered that Threonine synthase is essential fornormal rice blast pathogenicity, and can be used as a target for theidentification of antibiotics, preferably fungicides. Accordingly, thepresent invention provides methods for the identification of compoundsthat inhibit Threonine synthase expression or activity. The methods ofthe invention are useful for the identification of antibiotics,preferably fungicides.

BRIEF DESCRIPTION OF THE FIGURES

[0007]FIG. 1 shows the reaction performed by Threonine synthase (THR4)reaction. The Substrates/Products are O-phospho-L-homoserine and waterand the Products/Substrates are L-threonine and orthophosphate. Thefunction of the Threonine synthase enzyme is the interconversion ofO-phospho-L-homoserine and water to L-threonine and orthophosphate. Thisreaction is part of the L-threonine biosynthesis pathway.

[0008]FIG. 2 shows a digital image showing the effect of THR4 genedisruption on Magnaporthe grisea pathogenicity using whole plantinfection assays. Rice variety CO39 was inoculated with wild-type andthe transposon insertion strains, KO1-3 and KO1-22. Leaf segments wereimaged at five days post-inoculation.

[0009]FIG. 3. Verification of Gene Function by Analysis of NutritionalRequirements. Wild-type and transposon insertion strains, KO1-3 andKO1-22, were grown in (A) minimal media and (B) minimal media with theaddition of L-threonine, respectively. The x-axis shows time in days andthe y-axis shows turbidity measured at 490 nanometers and 750nanometers. The symbols represent wildtype (--♦--), transposon strainKO1-3 (--▪--), and transposon strain KO1-22 (--▴--).

DETAILED DESCRIPTION OF THE INVENTION

[0010] Unless otherwise indicated, the following terms are intended tohave the following meanings in interpreting the present invention.

[0011] The term “active against” in the context of compounds, agents, orcompositions having antibiotic activity indicates that the compoundexerts an effect on a particular target or targets which is deleteriousto the in vitro and/or in vivo growth of an organism having that targetor targets. In particular, a compound active against a gene exerts anaction on a target which affects an expression product of that gene.This does not necessarily mean that the compound acts directly on theexpression product of the gene, but instead indicates that the compoundaffects the expression product in a deleterious manner. Thus, the directtarget of the compound may be, for example, at an upstream componentwhich reduces transcription from the gene, resulting in a lower level ofexpression. Likewise, the compound may affect the level of translationof a polypeptide expression product, or may act on a downstreamcomponent of a biochemical pathway in which the expression product ofthe gene has a major biological role. Consequently, such a compound canbe said to be active against the gene, against the gene product, oragainst the related component either upstream or downstream of that geneor expression product. While the term “active against” encompasses abroad range of potential activities, it also implies some degree ofspecificity of target. Therefore, for example, a general protease is not“active against” a particular gene which produces a polypeptide product.In contrast, a compound which inhibits a particular enzyme is activeagainst that enzyme and against the gene which codes for that enzyme.

[0012] As used herein, the term “allele” refers to any of thealternative forms of a gene that may occur at a given locus.

[0013] The term “antibiotic” refers to any substance or compound thatwhen contacted with a living cell, organism, virus, or other entitycapable of replication, results in a reduction of growth, viability, orpathogenicity of that entity.

[0014] The term “binding” refers to a non-covalent or a covalentinteraction, preferably non-covalent, that holds two molecules together.For example, two such molecules could be an enzyme and an inhibitor ofthat enzyme. Non-covalent interactions include hydrogen bonding, ionicinteractions among charged groups, van der Waals interactions andhydrophobic interactions among nonpolar groups. One or more of theseinteractions can mediate the binding of two molecules to each other.

[0015] The term “biochemical pathway” or “pathway” refers to a connectedseries of biochemical reactions normally occurring in a cell, or morebroadly a cellular event such as cellular division or DNA replication.Typically, the steps in such a biochemical pathway act in a coordinatedfashion to produce a specific product or products or to produce someother particular biochemical action. Such a biochemical pathway requiresthe expression product of a gene if the absence of that expressionproduct either directly or indirectly prevents the completion of one ormore steps in that pathway, thereby preventing or significantly reducingthe production of one or more normal products or effects of thatpathway. Thus, an agent specifically inhibits such a biochemical pathwayrequiring the expression product of a particular gene if the presence ofthe agent stops or substantially reduces the completion of the series ofsteps in that pathway. Such an agent, may, but does not necessarily, actdirectly on the expression product of that particular gene.

[0016] As used herein, the term “cDNA” means complementarydeoxyribonucleic acid.

[0017] As used herein, the term “CoA” means coenzyme A.

[0018] As used herein, the term “conditional lethal” refers to amutation permitting growth and/or survival only under special growth orenvironmental conditions.

[0019] As used herein, the term “cosmid” refers to a hybrid vector, usedin gene cloning, that includes a cos site (from the lambdabacteriophage). It also contains drug resistance marker genes and otherplasmid genes. Cosmids are especially suitable for cloning large genesor multigene fragments.

[0020] As used herein, the term “dominant allele” refers to a dominantmutant allele in which a discernable mutant phenotype can be detectedwhen this mutation is present in an organism that also contains a wildtype (non-mutant), recessive allele, or other dominant allele.

[0021] As used herein, the term “DNA” means deoxyribonucleic acid.

[0022] As used herein, the term “ELISA” means enzyme-linkedimmunosorbent assay.

[0023] “Fungi” (singular: fungus) refers to whole fungi, fungal organsand tissues (e.g., asci, hyphae, pseudohyphae, rhizoid, sclerotia,sterigmata, spores, sporodochia, sporangia, synnemata, conidia,ascostroma, cleistothecia, mycelia, perithecia, basidia and the like),spores, fungal cells and the progeny thereof. Fungi are a group oforganisms (about 50,000 known species), including, but not limited to,mushrooms, mildews, moulds, yeasts, etc., comprising the kingdom Fungi.They can either exist as single cells or make up a multicellular bodycalled a mycelium, which consists of filaments known as hyphae. Mostfungal cells are multinucleate and have cell walls, composed chiefly ofchitin. Fungi exist primarily in damp situations on land and, because ofthe absence of chlorophyll and thus the inability to manufacture theirown food by photosynthesis, are either parasites on other organisms orsaprotrophs feeding on dead organic matter. The principal criteria usedin classification are the nature of the spores produced and the presenceor absence of cross walls within the hyphae. Fungi are distributedworldwide in terrestrial, freshwater, and marine habitats. Some live inthe soil. Many pathogenic fungi cause disease in animals and man or inplants, while some saprotrophs are destructive to timber, textiles, andother materials. Some fungi form associations with other organisms, mostnotably with algae to form lichens.

[0024] As used herein, the term “fungicide”, “antifungal”, or“antimycotic” refers to an antibiotic substance or compound that killsor suppresses the growth, viability, or pathogenicity of at least onefungus, fungal cell, fungal tissue or spore.

[0025] In the context of this disclosure, “gene” should be understood torefer to a unit of heredity. Each gene is composed of a linear chain ofdeoxyribonucleotides which can be referred to by the sequence ofnucleotides forming the chain. Thus, “sequence” is used to indicate boththe ordered listing of the nucleotides which form the chain, and thechain, itself, which has that sequence of nucleotides. (“Sequence” isused in the similar way in referring to RNA chains, linear chains madeof ribonucleotides). The gene may include regulatory and controlsequences, sequences which can be transcribed into an RNA molecule, andmay contain sequences with unknown function. The majority of the RNAtranscription products are messenger RNAs (mRNAs), which includesequences which are translated into polypeptides and may includesequences which are not translated. It should be recognized that smalldifferences in nucleotide sequence for the same gene can exist betweendifferent fungal strains, or even within a particular fungal strain,without altering the identity of the gene.

[0026] As used in this disclosure, the terms “growth” or “cell growth”of an organism refers to an increase in mass, density, or number ofcells of said organism. Some common methods for the measurement ofgrowth include the determination of the optical density of a cellsuspension, the counting of the number of cells in a fixed volume, thecounting of the number of cells by measurement of cell division, themeasurement of cellular mass or cellular volume, and the like.

[0027] As used in this disclosure, the term “growth conditionalphenotype” indicates that a fungal strain having such a phenotypeexhibits a significantly greater difference in growth rates in responseto a change in one or more of the culture parameters than an otherwisesimilar strain not having a growth conditional phenotype. Typically, agrowth conditional phenotype is described with respect to a singlegrowth culture parameter, such as temperature. Thus, a temperature (orheat-sensitive) mutant (i.e., a fungal strain having a heat-sensitivephenotype) exhibits significantly different growth, and preferably nogrowth, under non-permissive temperature conditions as compared togrowth under permissive conditions. In addition, such mutants preferablyalso show intermediate growth rates at intermediate, or semi-permissive,temperatures. Similar responses also result from the appropriate growthchanges for other types of growth conditional phenotypes.

[0028] As used herein, the term “H₂O” means water.

[0029] As used herein, the term “heterologous THR4 gene” means a gene,not derived from Magnaporthe grisea, and having: at least 50% sequenceidentity, preferably 60%, 70%, 80%, 90%, 95%, 99% sequence identity andeach integer unit of sequence identity from 50-100% in ascending orderto SEQ ID NO: 1 or SEQ ID NO: 2; or at least 10% of the activity of aMagnaporthe grisea Threonine synthase, preferably 25%, 50%, 75%, 90%,95%, 99% and each integer unit of activity from 10-100% in ascendingorder.

[0030] As used herein, the term “His-Tag” refers to an encodedpolypeptide consisting of multiple consecutive histidine amino acids.

[0031] As used herein, the term “HPLC” means high pressure liquidchromatography.

[0032] As used herein, the terms “hph”, “hygromycin Bphosphotransferase”, and “hygromycin resistance gene” refer to the E.coli hygromycin phosphotransferase gene or gene product.

[0033] As used herein, the term “hygromycin B” refers to anaminoglycosidic antibiotic, used for selection and maintenance ofeukaryotic cells containing the E. coli hygromycin resistance gene.

[0034] “Hypersensitive” refers to a phenotype in which cells are moresensitive to antibiotic compounds than are wild-type cells of similar oridentical genetic background.

[0035] “Hyposensitive” refers to a phenotype in which cells are lesssensitive to antibiotic compounds than are wild-type cells of similar oridentical genetic background.

[0036] As used herein, the term “imperfect state” refers to aclassification of a fungal organism having no demonstrable sexual lifestage.

[0037] The term “inhibitor”, as used herein, refers to a chemicalsubstance that inactivates the enzymatic activity of Threonine synthaseor substantially reduces the level of enzymatic activity, wherein“substantially” means a reduction at least as great as the standarddeviation for a measurement, preferably a reduction by 50%, morepreferably a reduction of at least one magnitude, i.e. to 10%. Theinhibitor may function by interacting directly with the enzyme, acofactor of the enzyme, the substrate of the enzyme, or any combinationthereof.

[0038] A polynucleotide may be “introduced” into a fungal cell by anymeans known to those of skill in the art, including transfection,transformation or transduction, transposable element, electroporation,particle bombardment, infection and the like. The introducedpolynucleotide may be maintained in the cell stably if it isincorporated into a non-chromosomal autonomous replicon or integratedinto the fungal chromosome. Alternatively, the introduced polynucleotidemay be present on an extra-chromosomal non-replicating vector and betransiently expressed or transiently active.

[0039] As used herein, the term “knockout” or “gene disruption” refersto the creation of organisms carrying a null mutation (a mutation inwhich there is no active gene product), a partial null mutation ormutations, or an alteration or alterations in gene regulation byinterrupting a DNA sequence through insertion of a foreign piece of DNA.Usually the foreign DNA encodes a selectable marker.

[0040] As used herein, the term “LB agar” means Luria's Broth agar.

[0041] The term “method of screening” means that the method is suitable,and is typically used, for testing for a particular property or effectin a large number of compounds. Typically, more than one compound istested simultaneously (as in a 96-well microtiter plate), and preferablysignificant portions of the procedure can be automated. “Method ofscreening” also refers to the determination of a set of differentproperties or effects of one compound simultaneously.

[0042] As used herein, the term “mRNA” means messenger ribonucleic acid.

[0043] As used herein, the term “mutant form” of a gene refers to a genewhich has been altered, either naturally or artificially, changing thebase sequence of the gene. The change in the base sequence may be ofseveral different types, including changes of one or more bases fordifferent bases, deletions, and/or insertions, such as by a transposon.By contrast, a normal form of a gene (wild type) is a form commonlyfound in natural populations of an organism. Commonly a single form of agene will predominate in natural populations. In general, such a gene issuitable as a normal form of a gene, however, other forms which providesimilar functional characteristics may also be used as a normal gene. Inparticular, a normal form of a gene does not confer a growth conditionalphenotype on the strain having that gene, while a mutant form of a genesuitable for use in these methods does provide such a growth conditionalphenotype.

[0044] As used herein, the term “Ni” refers to nickel.

[0045] As used herein, the term “Ni-NTA” refers to nickel sepharose.

[0046] As used herein, a “normal” form of a gene (wild type) is a formcommonly found in natural populations of an organism. Commonly a singleform of a gene will predominate in natural populations. In general, sucha gene is suitable as a normal form of a gene, however, other formswhich provide similar functional characteristics may also be used as anormal gene. In particular, a normal form of a gene does not confer agrowth conditional phenotype on the strain having that gene, while amutant form of a gene suitable for use in these methods does providesuch a growth conditional phenotype.

[0047] As used herein, the term “one form” of a gene is synonymous withthe term “gene”, and a “different form” of a gene refers to a gene thathas greater than 49% sequence identity and less than 100% sequenceidentity with said first form.

[0048] As used herein, the term “pathogenicity” refers to a capabilityof causing disease. The term is applied to parasitic microorganisms inrelation to their hosts.

[0049] As used herein, the term “PCR” means polymerase chain reaction.

[0050] The “percent (%) sequence identity” between two polynucleotide ortwo polypeptide sequences is determined according to the either theBLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W.Gish, et al. (1990) J Mol Biol 215: 403-10 (PMID: 2231712)) at theNational Center for Biotechnology or using Smith Waterman Alignment(Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147:195-7 (PMID:7265238)) as incorporated into GeneMatcher Plus™. It is understood thatfor the purposes of determining sequence identity when comparing a DNAsequence to an RNA sequence, a thymine nucleotide is equivalent to auracil nucleotide.

[0051] By “polypeptide” is meant a chain of at least two amino acidsjoined by peptide bonds. The chain may be linear, branched, circular orcombinations thereof. Preferably, polypeptides are from about 10 toabout 1000 amino acids in length, more preferably 10-50 amino acids inlength. The polypeptides may contain amino acid analogs and othermodifications, including, but not limited to glycosylated orphosphorylated residues.

[0052] As used herein, the term “proliferation” is synonymous to theterm “growth”.

[0053] As used herein, the term “reverse transcriptase-PCR” meansreverse transcription-polymerase chain reaction.

[0054] As used herein, the term “RNA” means ribonucleic acid.

[0055] As used herein, “semi-permissive conditions” are conditions inwhich the relevant culture parameter for a particular growth conditionalphenotype is intermediate between permissive conditions andnon-permissive conditions. Consequently, in semi-permissive conditionsan organism having a growth conditional phenotype will exhibit growthrates intermediate between those shown in permissive conditions andnon-permissive conditions. In general, such intermediate growth rate maybe due to a mutant cellular component which is partially functionalunder semi-permissive conditions, essentially fully functional underpermissive conditions, and is non-functional or has very low functionunder non-permissive conditions, where the level of function of thatcomponent is related to the growth rate of the organism. An intermediategrowth rate may also be a result of a nutrient substance or substancesthat are present in amounts not sufficient for optimal growth rates tobe achieved.

[0056] “Sensitivity phenotype” refers to a phenotype that exhibitseither hypersensitivity or hyposensitivity.

[0057] The term “specific binding” refers to an interaction betweenThreonine synthase and a molecule or compound, wherein the interactionis dependent upon the primary amino acid sequence and/or theconformation of Threonine synthase.

[0058] As used herein, the term “THR4” means a gene encoding Threoninesynthase activity, referring to an enzyme that catalyses theinterconversion of O-phospho-L-homoserine and water with L-threonine andorthophosphate, and may also be used to refer to the gene product.

[0059] As used herein, the terms “Threonine synthase” (EC 4.2.99.2) and“Threonine synthase polypeptide” are synonymous with “the THR4 geneproduct” and refer to an enzyme that catalyses the interconversion ofO-phospho-L-homoserine and water with L-threonine and orthophosphate.

[0060] As used herein, the term “TLC” means thin layer chromatography.

[0061] “Transform”, as used herein, refers to the introduction of apolynucleotide (single or double stranded DNA, RNA, or a combinationthereof) into a living cell by any means. Transformation may beaccomplished by a variety of methods, including, but not limited to,electroporation, polyethylene glycol mediated uptake, particlebombardment, agrotransformation, and the like. This process may resultin transient or stable expression of the transformed polynucleotide. By“stably transformed” is meant that the sequence of interest isintegrated into a replicon in the cell, such as a chromosome or episome.Transformed cells encompass not only the end product of a transformationprocess, but also the progeny thereof which retain the polynucleotide ofinterest.

[0062] For the purposes of the invention, “transgenic” refers to anycell, spore, tissue or part, that contains all or part of at least onerecombinant polynucleotide. In many cases, all or part of therecombinant polynucleotide is stably integrated into a chromosome orstable extra-chromosomal element, so that it is passed on to successivegenerations.

[0063] As used herein, the term “transposase” refers to an enzyme thatcatalyzes transposition. Preferred transposons are described in WO00/55346, PCT/US00/07317, and U.S. Ser. No. 09/658,859.

[0064] As used herein, the term “transposition” refers to a complexgenetic rearrangement process involving the movement or copying of apolynucleotide (transposon) from one location and insertion intoanother, often within or between a genome or genomes, or DNA constructssuch as plasmids, bacmids, and cosmids.

[0065] As used herein, the term “transposon” (also known as a“transposable element”, “transposable genetic element”, “mobileelement”, or “jumping gene”) refers to a mobile DNA element such asthose, for example, described in WO 00/55346, PCT/US00/07317, and U.S.Ser. No. 09/658,859. Transposons can disrupt gene expression or causedeletions and inversions, and hence affect both the genotype andphenotype of the organisms concerned. The mobility of transposableelements has long been used in genetic manipulation, to introduce genesor other information into the genome of certain model systems.

[0066] As used herein, the term “Tween 20” means sorbitanmono-9-octadecenoate poly(oxy-1,1-ethanediyl).

[0067] As used in this disclosure, the term “viability” of an organismrefers to the ability of an organism to demonstrate growth underconditions appropriate for said organism, or to demonstrate an activecellular function. Some examples of active cellular functions includerespiration as measured by gas evolution, secretion of proteins and/orother compounds, dye exclusion, mobility, dye oxidation, dye reduction,pigment production, changes in medium acidity, and the like.

[0068] The present inventors have discovered that disruption of the THR4gene and/or gene product inhibits the pathogenicity of Magnaporthegrisea. Thus, the inventors are the first to demonstrate that Threoninesynthase is a target for antibiotics, preferably antifungals.

[0069] Accordingly, the invention provides methods for identifyingcompounds that inhibit THR4 gene expression or biological activity ofits gene product(s). Such methods include ligand binding assays, assaysfor enzyme activity, cell-based assays, and assays for THR4 geneexpression. Any compound that is a ligand for Threonine synthase mayhave antibiotic activity. For the purposes of the invention, “ligand”refers to a molecule that will bind to a site on a polypeptide. Thecompounds identified by the methods of the invention are useful asantibiotics.

[0070] Thus, in one embodiment, the invention provides a method foridentifying a test compound as a candidate for an antibiotic,comprising:

[0071] a) contacting a Threonine synthase polypeptide with a testcompound; and

[0072] b) detecting the presence or absence of binding between said testcompound and said Threonine synthase polypeptide, wherein bindingindicates that said test compound is a candidate for an antibiotic.

[0073] The Threonine synthase protein may have the amino acid sequenceof a naturally occurring Threonine synthase found in a fungus, animal,plant, or microorganism, or may have an amino acid sequence derived froma naturally occurring sequence. Preferably the Threonine synthase is afungal Threonine synthase. The cDNA (SEQ ID NO: 1) encoding theThreonine synthase protein, the genomic DNA (SEQ ID NO: 2) encoding theM. grisea protein, and the polypeptide (SEQ ID NO: 3) can be foundherein.

[0074] In one aspect, the invention also provides for a polypeptideconsisting essentially of SEQ ID NO: 3. For the purposes of theinvention, a polypeptide consisting essentially of SEQ ID NO: 3 has atleast 80% sequence identity with SEQ ID NO: 3 and catalyses theinterconversion of O-phospho-L-homoserine and water with L-threonine andorthophosphate with at least 10% of the activity of SEQ ID NO: 3.Preferably, the polypeptide consisting essentially of SEQ ID NO: 3 hasat least 85% sequence identity with SEQ ID NO: 3, more preferably thesequence identity is at least 90%, most preferably the sequence identityis at least 95% or 97 or 99%, or any integer from 80-100% sequenceidentity in ascending order. And, preferably, the polypeptide consistingessentially of SEQ ID NO: 3 has at least 25%, at least 50%, at least 75%or at least 90% of the activity of M. grisea Threonine synthase, or anyinteger from 60-100% activity in ascending order.

[0075] By “fungal Threonine synthase” is meant an enzyme that can befound in at least one fungus, and which catalyzes the interconversion ofO-phospho-L-homoserine and water with L-threonine and orthophosphate.The Threonine synthase may be from any of the fungi, includingascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

[0076] In one embodiment, the Threonine synthase is a MagnaportheThreonine synthase. Magnaporthe species include, but are not limited to,Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea andMagnaporthe poae and the imperfect states of Magnaporthe in the genusPyricularia. Preferably, the Magnaporthe Threonine synthase is fromMagnaporthe grisea.

[0077] In various embodiments, the Threonine synthase can be fromPowdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea),White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum),Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot(Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), HoneyFungus (Armillaria gallica), Root rot (Armillaria luteobubalina),Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus(Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena),Apple-rotting Fungus (Penicillium expansum), Clubroot Disease(Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Rootpathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomycesgraminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromycesappendiculatus), Northern Leaf Spot (Cochliobolus carbonum), MiloDisease (Periconia circinata), Southern Corn Blight (Cochliobolusheterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe(Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), WheatHead Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusariumculmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black StemRust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), and thelike.

[0078] Fragments of a Threonine synthase polypeptide may be used in themethods of the invention, preferably if the fragments include an intactor nearly intact epitope that occurs on the biologically active wildtypeThreonine synthase. The fragments comprise at least 10 consecutive aminoacids of a Threonine synthase. Preferably, the fragment comprises atleast 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, or at least540 consecutive amino acids residues of a Threonine synthase. In oneembodiment, the fragment is from a Magnaporthe Threonine synthase.Preferably, the fragment contains an amino acid sequence conserved amongfungal Threonine synthases.

[0079] Polypeptides having at least 50% sequence identity with a fungalThreonine synthase are also useful in the methods of the invention.Preferably, the sequence identity is at least 60%, more preferably thesequence identity is at least 70%, most preferably the sequence identityis at least 80% or 90 or 95 or 99%, or any integer from 60-100% sequenceidentity in ascending order.

[0080] In addition, it is preferred that the polypeptide has at least10% of the activity of a fungal Threonine synthase. More preferably, thepolypeptide has at least 25%, at least 50%, at least 75% or at least 90%of the activity of a fungal Threonine synthase. Most preferably, thepolypeptide has at least 10%, at least 25%, at least 50%, at least 75%or at least 90% of the activity of the M. grisea Threonine synthaseprotein.

[0081] Thus, in another embodiment, the invention provides a method foridentifying a test compound as a candidate for a fungicide, comprising:

[0082] a) contacting a test compound with at least one polypeptideselected from the group consisting of: a polypeptide having at least tenconsecutive amino acids of a fungal Threonine synthase; a polypeptidehaving at least 50% sequence identity with a fungal Threonine synthase;and a polypeptide having at least 10% of the activity of a fungalThreonine synthase; and

[0083] b) detecting the presence and/or absence of binding between saidtest compound and said polypeptide, wherein binding indicates that saidtest compound is a candidate for an antibiotic.

[0084] Any technique for detecting the binding of a ligand to its targetmay be used in the methods of the invention. For example, the ligand andtarget are combined in a buffer. Many methods for detecting the bindingof a ligand to its target are known in the art, and include, but are notlimited to the detection of an immobilized ligand-target complex or thedetection of a change in the properties of a target when it is bound toa ligand. For example, in one embodiment, an array of immobilizedcandidate ligands is provided. The immobilized ligands are contactedwith a Threonine synthase protein or a fragment or variant thereof, theunbound protein is removed and the bound Threonine synthase is detected.In a preferred embodiment, bound Threonine synthase is detected using alabeled binding partner, such as a labeled antibody. In a variation ofthis assay, Threonine synthase is labeled prior to contacting theimmobilized candidate ligands. Preferred labels include fluorescent orradioactive moieties. Preferred detection methods include fluorescencecorrelation spectroscopy (FCS) and FCS-related confocal nanofluorimetricmethods.

[0085] Once a compound is identified as a candidate for an antibiotic,it can be tested for the ability to inhibit Threonine synthase enzymaticactivity. The compounds can be tested using either in vitro or cellbased assays. Alternatively, a compound can be tested by applying itdirectly to a fungus or fungal cell, or expressing it therein, andmonitoring the fungus or fungal cell for changes or decreases in growth,development, viability, pathogenicity, or alterations in geneexpression. Thus, in one embodiment, the invention provides a method fordetermining whether a compound identified as an antibiotic candidate byan above method has antifungal activity, further comprising: contactinga fungus or fungal cells with said antifungal candidate and detecting adecrease in the growth, viability, or pathogenicity of said fungus orfungal cells.

[0086] By decrease in growth, is meant that the antifungal candidatecauses at least a 10% decrease in the growth of the fungus or fungalcells, as compared to the growth of the fungus or fungal cells in theabsence of the antifungal candidate. By a decrease in viability is meantthat at least 20% of the fungal cells, or portion of the funguscontacted with the antifungal candidate are nonviable. Preferably, thegrowth or viability will be decreased by at least 40%. More preferably,the growth or viability will be decreased by at least 50%, 75% or atleast 90% or more. Methods for measuring fungal growth and cellviability are known to those skilled in the art. By decrease inpathogenicity, is meant that the anti fungal candidate causes at least a10% decrease in the disease caused by contact of the fungal pathogenwith its host, as compared to the disease caused in the absence of theantifungal candidate. Preferably, the disease will be decreased by atleast 40%. More preferably, the disease will be decreased by at least50%, 75% or at least 90% or more. Methods for measuring fungal diseaseare well known to those skilled in the art, and include such metrics aslesion formation, lesion size, sporulation, respiratory failure, and/ordeath.

[0087] The ability of a compound to inhibit Threonine synthase activitycan be detected using in vitro enzymatic assays in which thedisappearance of a substrate or the appearance of a product is directlyor indirectly detected. Threonine synthase catalyzes the irreversible orreversible reaction O-phospho-L-homoserine and water=L-threonine andorthophosphate (see FIG. 1). Methods for detection ofO-phospho-L-homoserine, L-threonine, orthophosphate, and water, includespectrophotometry, mass spectroscopy, thin layer chromatography (TLC)and reverse phase HPLC.

[0088] Thus, the invention provides a method for identifying a testcompound as a candidate for an antibiotic, comprising:

[0089] a) contacting O-phospho-L-homoserine and water with a Threoninesynthase;

[0090] b) contacting O-phospho-L-homoserine and water with Threoninesynthase and said test compound; and

[0091] c) determining the change in concentration for at least one ofthe following: O-phospho-L-homoserine, L-threonine, orthophosphate, andwater, wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0092] An additional method is provided by the invention for identifyinga test compound as a candidate for an antibiotic, comprising:

[0093] a) contacting L-threonine and orthophosphate with a Threoninesynthase;

[0094] b) contacting L-threonine and orthophosphate with a Threoninesynthase and a test compound; and

[0095] c) determining the change in concentration for at least one ofthe following: O-phospho-L-homoserine, L-threonine, orthophosphate, andwater, wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0096] Enzymatically active fragments of a fungal Threonine synthase arealso useful in the methods of the invention. For example, anenzymatically active polypeptide comprising at least 100 consecutiveamino acid residues of a fungal Threonine synthase may be used in themethods of the invention. In addition, an enzymatically activepolypeptide having at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98%sequence identity with a fungal Threonine synthase may be used in themethods of the invention. Most preferably, the polypeptide has at least50% sequence identity with a fungal Threonine synthase and at least 10%,25%, 75% or at least 90% of the activity thereof.

[0097] Thus, the invention provides a method for identifying a testcompound as a candidate for an antibiotic, comprising:

[0098] a) contacting O-phospho-L-homoserine and water with a polypeptideselected from the group consisting of: a polypeptide having at least 50%sequence identity with a Threonine synthase, and a polypeptide having atleast 50% sequence identity with a Threonine synthase and having atleast 10% of the activity thereof, and a polypeptide comprising at least100 consecutive amino acids of a Threonine synthase;

[0099] b) contacting O-phospho-L-homoserine and water with saidpolypeptide and a test compound; and

[0100] c) determining the change in concentration for at least one ofthe following: O-phospho-L-homoserine, L-threonine, orthophosphate, andwater, wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0101] An additional method is provided by the invention for identifyinga test compound as a candidate for an antibiotic, comprising:

[0102] a) contacting L-threonine and orthophosphate with a polypeptideselected from the group consisting of: a polypeptide having at least 50%sequence identity with a Threonine synthase, and a polypeptide having atleast 50% sequence identity with a Threonine synthase and at least 10%of the activity thereof, and a polypeptide comprising at least 100consecutive amino acids of a Threonine synthase;

[0103] b) contacting L-threonine and orthophosphate, with saidpolypeptide and a test compound; and

[0104] c) determining the change in concentration for at least one ofthe following, O-phospho-L-homoserine, L-threonine, orthophosphate, andwater, wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0105] For the in vitro enzymatic assays, Threonine synthase protein andderivatives thereof may be purified from a fungus or may berecombinantly produced in and purified from an archael, bacterial,fungal, or other eukaryotic cell culture. Preferably these proteins areproduced using an E. coli, yeast, or filamentous fungal expressionsystem. Methods for the purification of Threonine synthase may bedescribed in Malumbres et al. (1994) Appl Environ Microbiol 60: 2209-19(PMID: 8074505). Other methods for the purification of Threoninesynthase proteins and polypeptides are known to those skilled in theart.

[0106] As an alternative to in vitro assays, the invention also providescell based assays. In one embodiment, the invention provides a methodfor identifying a test compound as a candidate for an antibiotic,comprising:

[0107] a) measuring the expression of a Threonine synthase in a cell,cells, tissue, or an organism in the absence of a test compound;

[0108] b) contacting said cell, cells, tissue, or organism with saidtest compound and measuring the expression of said Threonine synthase insaid cell, cells, tissue, or organism; and

[0109] c) comparing the expression of Threonine synthase in steps (a)and (b), wherein a lower expression in the presence of said testcompound indicates that said compound is a candidate for an antibiotic.

[0110] Expression of Threonine synthase can be measured by detecting theTHR4 primary transcript or mRNA, Threonine synthase polypeptide, orThreonine synthase enzymatic activity. Methods for detecting theexpression of RNA and proteins are known to those skilled in the art.See, for example, Current Protocols in Molecular Biology Ausubel et al.,eds., Greene Publishing and Wiley-Interscience, New York, 1995. Themethod of detection is not critical to the invention. Methods fordetecting THR4 RNA include, but are not limited to amplification assayssuch as quantitative reverse transcriptase-PCR, and/or hybridizationassays such as Northern analysis, dot blots, slot blots, in-situhybridization, transcriptional fusions using a THR4 promoter fused to areporter gene, DNA assays, and microarray assays.

[0111] Methods for detecting protein expression include, but are notlimited to, immunodetection methods such as Western blots, ELISA assays,polyacrylamide gel electrophoresis, mass spectroscopy, and enzymaticassays. Also, any reporter gene system may be used to detect THR4protein expression. For detection using gene reporter systems, apolynucleotide encoding a reporter protein is fused in frame with THR4,so as to produce a chimeric polypeptide. Methods for using reportersystems are known to those skilled in the art.

[0112] Chemicals, compounds or compositions identified by the abovemethods as modulators, preferably inhibitors, of THR4 expression oractivity can then be used to control fungal growth. Diseases such asrusts, mildews, and blights spread rapidly once established. Fungicidesare thus routinely applied to growing and stored crops as a preventivemeasure, generally as foliar sprays or seed dressings. For example,compounds that inhibit fungal growth can be applied to a fungus orexpressed in a fungus, in order to prevent fungal growth. Thus, theinvention provides a method for inhibiting fungal growth, comprisingcontacting a fungus with a compound identified by the methods of theinvention as having antifungal activity.

[0113] Antifungals and antifungal inhibitor candidates identified by themethods of the invention can be used to control the growth of undesiredfungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota,and lichens.

[0114] Examples of undesired fungi include, but are not limited toPowdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea),White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum),Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot(Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), HoneyFungus (Armillaria gallica), Root rot (Armillaria luteobubalina),Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus(Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena),Apple-rotting Fungus (Penicillium expansum), Clubroot Disease(Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Rootpathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomycesgraminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromycesappendiculatus), Northern Leaf Spot (Cochliobolus carbonum), MiloDisease (Periconia circinata), Southern Corn Blight (Cochliobolusheterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe(Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), WheatHead Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusariumculmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black StemRust (Puccinia graminis), White mold (Sclerotinia sclerotiorum),diseases of animals such as infections of lungs, blood, brain, skin,scalp, nails or other tissues (Aspergillus fumigatus Aspergillus sp.Fusraium sp., Trichophyton sp., Epidermophyton sp., and Microsporum sp.,and the like).

[0115] Also provided is a method of screening for an antibiotic bydetermining whether a test compound is active against the geneidentified (SEQ ID NO: 1 or SEQ ID NO: 2), its gene product (SEQ ID NO:3), or the biochemical pathway or pathways on which it functions.

[0116] In one particular embodiment, the method is performed byproviding an organism having a first form of the gene corresponding toeither SEQ ID NO: 1 or SEQ ID NO: 2, either a normal form, a mutantform, a homologue, or a heterologous THR4 gene that performs a similarfunction as THR4. The first form of THR4 may or may not confer a growthconditional phenotype, i.e., a L-threonine requiring phenotype, and/or ahypersensitivity or hyposensitivity phenotype on the organism havingthat altered form. In one particular embodiment a mutant form contains atransposon insertion. A comparison organism having a second form of aTHR4, different from the first form of the gene is also provided, andthe two organisms are separately contacted with a test compound. Thegrowth of the two organisms in the presence of the test compound is thencompared.

[0117] Thus, in one embodiment, the invention provides a method foridentifying a test compound as a candidate for an antibiotic,comprising:

[0118] a) providing cells having one form of a Threonine synthase gene,and providing comparison cells having a different form of a Threoninesynthase gene; and

[0119] b) contacting said cells and said comparison cells with a testcompound and determining the growth of said cells and said comparisoncells in the presence of the test compound, wherein a difference ingrowth between said cells and said comparison cells in the presence ofsaid test compound indicates that said test compound is a candidate foran antibiotic.

[0120] It is recognized in the art that the optional determination ofthe growth of said first organism and said comparison second organism inthe absence of any test compounds may be performed to control for anyinherent differences in growth as a result of the different genes. It isalso recognized that any combination of two different forms of a THR4gene, including normal genes, mutant genes, homologues, and functionalhomologues may be used in this method. Growth and/or proliferation of anorganism is measured by methods well known in the art such as opticaldensity measurements, and the like. In a preferred embodiment theorganism is Magnaporthe grisea.

[0121] Conditional lethal mutants may identify particular biochemicaland/or genetic pathways given that at least one identified target geneis present in that pathway. Knowledge of these pathways allows for thescreening of test compounds as candidates for antibiotics as inhibitorsof the substrates, products and enzymes of the pathway. Pathways knownin the art may be found at the Kyoto Encyclopedia of Genes and Genomesand in standard biochemistry texts (Lehninger, A., D. Nelson, et al.(1993) Principles of Biochemistry, New York, Worth Publishers).

[0122] Thus, in one embodiment, the invention provides a method forscreening for test compounds acting against the biochemical and/orgenetic pathway or pathways in which THR4 functions, comprising:

[0123] a) providing cells having one form of a gene in the L-threoninebiochemical and/or genetic pathway and providing comparison cells havinga different form of said gene;

[0124] b) contacting said cells and said comparison cells with a testcompound; and

[0125] c) determining the growth of said cells and said comparison cellsin the presence of said test compound, wherein a difference in growthbetween said cells and said comparison cells in the presence of saidtest compound indicates that said test compound is a candidate for anantibiotic.

[0126] The use of multi-well plates for screening is a format thatreadily accommodates multiple different assays to characterize variouscompounds, concentrations of compounds, and fungal strains in varyingcombinations and formats. Certain testing parameters for the screeningmethod can significantly affect the identification of growth inhibitors,and thus can be manipulated to optimize screening efficiency and/orreliability. Notable among these factors are variable sensitivities ofdifferent mutants, increasing hypersensitivity with increasingly lesspermissive conditions, an apparent increase in hypersensitivity withincreasing compound concentration, and other factors known to those inthe art.

[0127] Conditional lethal mutants may identify particular biochemicaland/or genetic pathways given that at least one identified target geneis present in that pathway. Knowledge of these pathways allows for thescreening of test compounds as candidates for antibiotics. Pathwaysknown in the art may be found at the Kyoto Encyclopedia of Genes andGenomes and in standard biochemistry texts (Lehninger et al. (1993)Principles of Biochemistry).

[0128] Thus, in one embodiment, the invention provides a method forscreening for test compounds acting against the biochemical and/orgenetic pathway or pathways in which THR4 functions, comprising:

[0129] (a) providing paired growth media comprising a first medium and asecond medium, wherein said second medium contains a higher level ofL-threonine than said first medium;

[0130] (b) contacting an organism with a test compound;

[0131] (c) inoculating said first and said second media with saidorganism; and

[0132] (d) determining the growth of said organism, wherein a differencein growth of the organism between said first and said second mediaindicates that said test compound is a candidate for an antibiotic.

[0133] It is recognized in the art that determination of the growth ofsaid organism in the paired media in the absence of any test compoundsmay be performed to control for any inherent differences in growth as aresult of the different media. Growth and/or proliferation of anorganism is measured by methods well known in the art such as opticaldensity measurements, and the like. In a preferred embodiment, theorganism is Magnaporthe grisea.

EXPERIMENTAL Example 1 Construction of Plasmids with a TransposonContaining a Selectable Marker

[0134] Construction of Sif transposon: Sif was constructed using theGPS3 vector from the GPS-M mutagenesis system from New England Biolabs,Inc. (Beverly, Mass.) as a backbone. This system is based on thebacterial transposon Tn7. The following manipulations were done to GPS3according to Sambrook et al (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press. The kanamycin resistancegene (npt) contained between the Tn7 arms was removed by EcoRVdigestion. The bacterial hygromycin B phosphotransferase (hph) gene(Gritz and Davies (1983) Gene 25: 179-88 (PMID: 6319235)) under controlof the Aspergillus nidulans trpC promoter and terminator (Mullaney etal. (1985) Mol Gen Genet 199: 37-45 (PMID: 3158796)) was cloned by aHpaI/EcoRV blunt ligation into the Tn7 arms of the GPS3 vector yieldingpSif1. Excision of the ampicillin resistance gene (bla) from pSif1 wasachieved by cutting pSif1 with XmnI and BglI followed by a T4 DNApolymerase treatment to remove the 3′ overhangs left by the BglIdigestion and religation of the plasmid to yield pSif. Top 10F′electrocompetent E. coli cells (Invitrogen) were transformed withligation mixture according to manufacturer's recommendations.Transformants containing the Sif transposon were selected on LB agar(Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual)containing 50 ug/ml of hygromycin B (Sigma Chem. Co., St. Louis, Mo.).

Example 2 Construction of a Fungal Cosmid Library

[0135] Cosmid libraries were constructed in the pcosKA5 vector (Hamer etal. (2001) Proc Natl Acad Sci USA 98: 5110-15 (PMID: 11296265)) asdescribed in Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual. Cosmid libraries were quality checked by pulsed-field gelelectrophoresis, restriction digestion analysis, and PCR identificationof single genes.

Example 3 Construction of Cosmids with Transposon Insertion into FungalGenes

[0136] Sif Transposition into a Cosmid: Transposition of Sif into thecosmid framework was carried out as described by the GPS-M mutagenesissystem (New England Biolabs, Inc.). Briefly, 2 ul of the 10X GPS buffer,70 ng of supercoiled pSIF, 8-12 ug of target cosmid DNA were mixed andtaken to a final volume of 20 ul with water. 1 ul of transposase(TnsABC) was added to the assembly reaction and incubated for 10 minutesat 37° C. After the assembly reaction, 1 ul of start solution was addedto the tube, mixed well and incubated for 1 hour at 37° C. followed byheat inactivation of the proteins at 75° C. for 10 min. Destruction ofthe remaining untransposed pSif was done by PISceI digestion at 37° C.for 2 hours followed by 10 min incubation at 75° C. to inactivate theproteins. Transformation of Top10 F′ electrocompetent cells (Invitrogen)was done according to manufacturers recommendations. Sif-containingcosmid transformants were selected by growth on LB agar platescontaining 50 ug/ml of hygromycin B (Sigma Chem. Co.) and 100 ug/ml ofAmpicillin (Sigma Chem. Co.).

Example 4 High Throughput Preparation and Verification of TransposonInsertion into the M. grisea THR4 Gene

[0137]E. coli strains containing cosmids with transposon insertions werepicked to 96 well growth blocks (Beckman Co.) containing 1.5 ml of TB(Terrific Broth, Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press) supplemented with 50 ug/mlof ampicillin. Blocks were incubated with shaking at 37° C. overnight.E. coli cells were pelleted by centrifugation and cosmids were isolatedby a modified alkaline lysis method (Marra et al. (1997) Genome Res 7:1072-84 (PMID: 9371743)). DNA quality was checked by electrophoresis onagarose gels. Cosmids were sequenced using primers from the ends of eachtransposon and commercial dideoxy sequencing kits (Big Dye Terminators,Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNAsequencer (Perkin Elmer Co.).

[0138] DNA sequences adjacent to the site of the insertion werecollected and used to search DNA and protein databases using the BLASTalgorithms (Altschul et al. (1997) Nucleic Acids Res 25: 3389-3402(PMID: 9254694)). A single insertion of SIF into the Magnaporthe griseaTHR4 gene was chosen for further analysis. This construct was designatedcpgmra0012020a04 and it contains the SIF transposon approximatelybetween amino acids 314 and 315 relative to the Schizosaccharomycespombe homologue ThrC (total length: 514 amino acids, GENBANK: 2501152).

Example 5 Preparation of THR4 Cosmid DNA and Transformation ofMagnaporthe grisea

[0139] Cosmid DNA from the THR4 transposon tagged cosmid clone wasprepared using QIAGEN Plasmid Maxi Kit (QIAGEN), and digested by PI-PspI(New England Biolabs, Inc.). Fungal electro-transformation was performedessentially as described (Wu et al. (1997) MPMI 10: 700-708). Briefly,M. grisea strain Guy 11 was grown in complete liquid media (Talbot etal. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) shaking at 120 rpmfor 3 days at 25° C. in the dark. Mycelia was harvested and washed withsterile H₂O and digested with 4 mg/ml beta-glucanase (InterSpex) for 4-6hours to generate protoplasts. Protoplasts were collected bycentrifugation and resuspended in 20% sucrose at the concentration of2×10⁸ protoplasts/ml. 50 ul protoplast suspension was mixed with 10-20ug of the cosmid DNA and pulsed using Gene Pulser II (BioRad) set withthe following parameters: resistance 200 ohm, capacitance 25 uF, voltage0.6 kV. Transformed protoplasts were regenerated in complete agar media(C M, Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) withthe addition of 20% sucrose for one day, then overlayed with CM agarmedia containing hygromycin B (250 ug/ml) to select transformants.Transformants were screened for homologous recombination events in thetarget gene by PCR (Hamer et al. (2001) Proc Natl Acad Sci USA 98:5110-15 (PMID: 11296265)). Two independent strains were identified andare hereby referred to as KO1-3 and KO1-22, respectively.

Example 6 Effect of Transposon Insertion on Magnaporthe Pathogenicity

[0140] The target fungal strains, KO1-3 and KO1-22, obtained in Example5 and the wild type strain, Guy11, were subjected to a pathogenicityassay to observe infection over a 1-week period. Rice infection assayswere performed using Indian rice cultivar CO39 essentially as describedin Valent et al. ((1991) Genetics 127: 87-101 (PMID: 2016048)). Allthree strains were grown for spore production on complete agar media.Spores were harvested and the concentration of spores adjusted for wholeplant inoculations. Two-week-old seedlings of cultivar CO39 were sprayedwith 12 ml of conidial suspension (5×10⁴ conidia per ml in 0.01%Tween-20 (Polyoxyethylensorbitan monolaureate) solution). The inoculatedplants were incubated in a dew chamber at 27° C. in the dark for 36hours, and transferred to a growth chamber (27° C. 12 hours/21° C. 12hours 70% humidity) for an additional 5.5 days. Leaf samples were takenat 3, 5, and 7 days post-inoculation and examined for signs ofsuccessful infection (i.e. lesions). FIG. 2 shows the effects of THR4gene disruption on Magnaporthe infection at five days post-inoculation.

Example 7 Verification of THR4 Gene Function by Analysis of NutritionalRequirements

[0141] The fungal strains, KO1-3 and KO1-22, containing the THR4disrupted gene obtained in Example 5 were analyzed for their nutritionalrequirement for L-threonine using the PM5 phenotype microarray fromBiolog, Inc. (Hayward, Calif.). The PM5 plate tests for the auxotrophicrequirement for 94 different metabolites. The inoculating fluid consistsof 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5 mM NaNO₃, 6.7 mMKCl, 3.5 mM Na₂SO₄, 11 mM KH₂PO₄, 0.01% p-iodonitrotetrazolium violet,0.1 mM MgCl₂, 1.0 mM CaCl₂ and trace elements, pH adjusted to 6.0 withNaOH. Final concentrations of trace elements are: 7.6 μM ZnCl₂, 2.5 μMMnCl₂.4H₂O, 1.8 μM FeCl₂.4H₂O, 0.71 μM CoCl₂.6H₂O, 0.64 μM CuCl₂.2H₂O,0.62 μM Na₂MoO₄, 18 μM H₃BO₃. Spores for each strain were harvested intothe inoculating fluid. The spore concentrations were adjusted to 2×10⁵spores/ml. 100 μl of spore suspension were deposited into each well ofthe microtiter plates. The plates were incubated at 25° C. for 7 days.Optical density (OD) measurements at 490 nm and 750 nm were taken daily.The OD₄₉₀ measures the extent of tetrazolium dye reduction and the levelof growth, and OD₇₅₀ measures growth only. Turbidity=OD₄₉₀+OD₇₅₀. Dataconfirming the annotated gene function is presented as a graph ofTurbidity vs. Time showing both the mutant fungi and the wild-typecontrol in the absence (FIG. 3A) and presence (FIG. 3B) of L-threonine.

Example 8 Cloning and Expression Strategies, Extraction and Purificationof Threonine Synthase Protein

[0142] The following protocol may be employed to obtain a purifiedThreonine synthase protein.

[0143] Cloning and Expression Strategies:

[0144] A THR4 cDNA gene can be cloned into E. coli (pETvectors-Novagen), Baculovirus (Pharmingen) and Yeast (Invitrogen)expression vectors containing His/fusion protein tags, and theexpression of recombinant protein can be evaluated by SDS-PAGE andWestern blot analysis.

[0145] Extraction:

[0146] Extract recombinant protein from 250 ml cell pellet in 3 ml ofextraction buffer by sonicating 6 times, with 6 sec pulses at 4° C.Centrifuge extract at 15000×g for 10 min and collect supernatant. Assessbiological activity of the recombinant protein by activity assay.

[0147] Purification:

[0148] Purify recombinant protein by Ni-NTA affinity chromatography(Qiagen). Purification protocol: perform all steps at 4° C.:

[0149] Use 3 ml Ni-beads (Qiagen)

[0150] Equilibrate column with the buffer

[0151] Load protein extract

[0152] Wash with the equilibration buffer

[0153] Elute bound protein with 0.5 M imidazole

Example 9

[0154] Assays for Testing Binding of Test Compounds to ThreonineSynthase

[0155] The following protocol may be employed to identify test compoundsthat bind to the Threonine synthase protein.

[0156] Purified full-length Threonine synthase polypeptide with aHis/fusion protein tag (Example 8) is bound to a HisGrab™ Nickel CoatedPlate (Pierce, Rockford, Ill.) following manufacturer's instructions.

[0157] Buffer conditions are optimized (e.g. ionic strength or pH, Ramosand Calderon (1994) FEBS Lett 351: 357-9 (PMID: 8082795)) for binding ofradiolabeled O-phospho-L-homoserine (Gening et al. (1994) Biokhimiia 59:1238-44 (PMID: 7819407)) to the bound Threonine synthase.

[0158] Screening of test compounds is performed by adding test compoundand radiolabeled O-phospho-L-homoserine (Gening et al. (1994) Biokhimiia59: 1238-44 (PMID: 7819407)) to the wells of the HisGrab™ platecontaining bound Threonine synthase.

[0159] The wells are washed to remove excess labeled ligand andscintillation fluid (Scintiverse®, Fisher Scientific) is added to eachwell.

[0160] The plates are read in a microplate scintillation counter.

[0161] Candidate compounds are identified as wells with lowerradioactivity as compared to control wells with no test compound added.

[0162] Additionally, a purified polypeptide comprising 10-50 amino acidsfrom the M. grisea Threonine synthase is screened in the same way. Apolypeptide comprising 10-50 amino acids is generated by subcloning aportion of the THR4 gene into a protein expression vector that adds aHis-Tag when expressed (see Example 8). Oligonucleotide primers aredesigned to amplify a portion of the THR4 gene using the polymerasechain reaction amplification method. The DNA fragment encoding apolypeptide of 10-50 amino acids is cloned into an expression vector,expressed in a host organism and purified as described in Example 8above.

[0163] Test compounds that bind THR4 are further tested for antibioticactivity. M. grisea is grown as described for spore production onoatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID:8312740)). Spores are harvested into minimal media (Talbot et al (1993)Plant Cell 5: 1575-1590 (PMID: 8312740)) to a concentration of 2×10⁵spores/ml and the culture is divided. The test compound is added to oneculture to a final concentration of 20-100 μg/ml. Solvent only is addedto the second culture. The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. The growthcurves of the solvent control sample and the test compound sample arecompared. A test compound is an antibiotic candidate if the growth ofthe culture containing the test compound is less than the growth of thecontrol culture.

Example 10 Assays for Testing Inhibitors or Candidates for Inhibition ofThreonine Synthase Activity

[0164] The enzymatic activity of Threonine synthase is determined in thepresence and absence of candidate compounds in a suitable reactionmixture, such as described by Ramos and Calderon (1994) FEBS Lett 351:357-9 (PMID: 8082795). Candidate compounds are identified when adecrease in products or a lack of decrease in substrates is detectedwith the reaction proceeding in either direction.

[0165] Additionally, the enzymatic activity of a polypeptide comprising10-50 amino acids from the M. grisea Threonine synthase is determined inthe presence and absence of candidate compounds in a suitable reactionmixture, such as described by Ramos and Calderon (1994) FEBS Lett 351:357-9 (PMID: 8082795). A polypeptide comprising 10-50 amino acids isgenerated by subcloning a portion of the THR4 gene into a proteinexpression vector that adds a His-Tag when expressed (see Example 8).Oligonucleotide primers are designed to amplify a portion of the THR4gene using polymerase chain reaction amplification method. The DNAfragment encoding a polypeptide of 10-50 amino acids is cloned into anexpression vector, expressed and purified as described in Example 8above.

[0166] Test compounds identified as inhibitors of THR4 activity arefurther tested for antibiotic activity. Magnaporthe grisea fungal cellsare grown under standard fungal growth conditions that are well knownand described in the art. M. grisea is grown as described for sporeproduction on oatmeal agar media (Talbot et al. (1993) Plant Cell 5:1575-1590 (PMID: 8312740)). Spores are harvested into minimal media(Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) to aconcentration of 2×10⁵ spores/ml and the culture is divided. The testcompound is added to one culture to a final concentration of 20-100μg/ml. Solvent only is added to the second culture. The plates areincubated at 25° C. for seven days and optical density measurements at590 nm are taken daily. The growth curves of the solvent control sampleand the test compound sample are compared. A test compound is anantibiotic candidate if the growth of the culture containing the testcompound is less than the growth of the control culture.

Example 11 Assays for Testing Compounds for Alteration of ThreonineSynthase Gene Expression

[0167]Magnaporthe grisea fungal cells are grown under standard fungalgrowth conditions that are well known and described in the art.Wild-type M. grisea spores are harvested from cultures grown on completeagar or oatmeal agar media after growth for 10-13 days in the light at25° C. using a moistened cotton swab. The concentration of spores isdetermined using a hemacytometer and spore suspensions are prepared in aminimal growth medium to a concentration of 2×10⁵ spores per ml. 25 mlcultures are prepared to which test compounds will be added at variousconcentrations. A culture with no test compound present is included as acontrol. The cultures are incubated at 25° C. for 3 days after whichtest compound or solvent only control is added. The cultures areincubated an additional 18 hours. Fungal mycelia is harvested byfiltration through Miracloth (CalBiochem®, La Jolla, Calif.), washedwith water and frozen in liquid nitrogen. Total RNA is extracted withTRIZOL® Reagent using the methods provided by the manufacturer (LifeTechnologies, Rockville, Md.). Expression is analyzed by Northernanalysis of the RNA samples as described (Sambrook et al. (1989)Molecular Cloning, a Laboratory Manual, Cold Spring Harbor LaboratoryPress) using a radiolabeled fragment of the THR4 gene as a probe. Testcompounds resulting in a reduced level of THR4 mRNA relative to theuntreated control sample are identified as candidate antibioticcompounds.

Example 12 In vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of Threonine Synthase with No Activity

[0168]Magnaporthe grisea fungal cells containing a mutant form of theTHR4 gene which abolishes enzyme activity, such as a gene containing atransposon insertion (see Examples 4 and 5), are grown under standardfungal growth conditions that are well known and described in the art.Magnaporthe grisea spores are harvested from cultures grown on completeagar medium containing 4 mM L-threonine (Sigma-Aldrich Co.) after growthfor 10-13 days in the light at 25° C. using a moistened cotton swab. Theconcentration of spores is determined using a hemacytometer and sporesuspensions are prepared in a minimal growth medium containing 100 μML-threonine to a concentration of 2×10⁵ spores per ml. Approximately4×10⁴ spores are added to each well of 96-well plates to which a testcompound is added (at varying concentrations). The total volume in eachwell is 200 μl. Wells with no test compound present (growth control),and wells without cells are included as controls (negative control). Theplates are incubated at 25° C. for seven days and optical densitymeasurements at 590 nm are taken daily. Wild type cells are screenedunder the same conditions. The effect of each compound on the mutant andwild-type fungal strains is measured against the growth control and thepercent of inhibition is calculated as the OD₅₉₀ (fungal strain plustest compound)/OD₅₉₀ (growth control)×100. The percent of growthinhibition as a result of a test compound on a fungal strain and that onthe wild type cells are compared. Compounds that show differentialgrowth inhibition between the mutant and the wild type are identified aspotential antifungal compounds. Similar protocols may be found in Kirschand DiDomenico ((1994) Biotechnology 26:177-221 (PMID: 7749303)).

Example 13 In vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of Threonine Synthase with ReducedActivity

[0169]Magnaporthe grisea fungal cells containing a mutant form of theTHR4 gene, such as a promoter truncation that reduces expression, aregrown under standard fungal growth conditions that are well known anddescribed in the art. A promoter truncation is made by deleting aportion of the promoter upstream of the transcription start site usingstandard molecular biology techniques that are well known and describedin the art (Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea sporesare harvested from cultures grown on complete agar medium containing 4mM L-threonine (Sigma-Aldrich Co.) after growth for 10-13 days in thelight at 25° C. using a moistened cotton swab. The concentration ofspores is determined using a hemacytometer and spore suspensions areprepared in a minimal growth medium to a concentration of 2×10⁵ sporesper ml. Approximately 4×10⁴ spores are added to each well of 96-wellplates to which a test compound is added (at varying concentrations).The total volume in each well is 200 μl. Wells with no test compoundpresent (growth control), and wells without cells are included ascontrols (negative control). The plates are incubated at 25° C. forseven days and optical density measurements at 590 nm are taken daily.Wild type cells are screened under the same conditions. The effect ofeach compound on the mutant and wild-type fungal strains is measuredagainst the growth control and the percent of inhibition is calculatedas the OD₅₉₀ (fungal strain plus test compound)/OD₅₉₀ (growthcontrol)×100. The percent of growth inhibition as a result of a testcompound on a fungal strain and that on the wild-type cells arecompared. Compounds that show differential growth inhibition between themutant and the wild type are identified as potential antifungalcompounds. Similar protocols may be found in Kirsch and DiDomenico((1994) Biotechnology 26: 177-221).

Example 14 In vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of a L-threonine Biosynthetic Gene withNo Activity

[0170]Magnaporthe grisea fungal cells containing a mutant form of a genein the L-threonine biosynthetic pathway (e.g. Homoserine kinase (E.C.2.7.1.39)) are grown under standard fungal growth conditions that arewell known and described in the art. Magnaporthe grisea spores areharvested from cultures grown on complete agar medium containing 4 mML-threonine (Sigma-Aldrich Co.) after growth for 10-13 days in the lightat 25° C. using a moistened cotton swab. The concentration of spores isdetermined using a hemacytometer and spore suspensions are prepared in aminimal growth medium containing 100 μM L-threonine to a concentrationof 2×10⁵ spores per ml. Approximately 4×10⁴ spores or cells areharvested and added to each well of 96-well plates to which growth mediais added in addition to an amount of test compound (at varyingconcentrations). The total volume in each well is 200 μl. Wells with notest compound present, and wells without cells are included as controls.The plates are incubated at 25° C. for seven days and optical densitymeasurements at 590 nm are taken daily. Wild type cells are screenedunder the same conditions. The effect of each compound on the mutant andwild-type fungal strains is measured against the growth control and thepercent of inhibition is calculated as the OD₅₉₀ (fungal strain plustest compound)/OD₅₉₀ (growth control)×100. The percent of growthinhibition as a result of a test compound on a fungal strain and that onthe wild type cells are compared. Compounds that show differentialgrowth inhibition between the mutant and the wild-type are identified aspotential antifungal compounds. Similar protocols may be found in Kirschand DiDomenico ((1994) Biotechnology 26: 177-221).

Example 15 In vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of a L-threonine Biosynthetic Gene withReduced Activity

[0171]Magnaporthe grisea fungal cells containing a mutant form of a genein the L-threonine biosynthetic pathway (e.g. Homoserine kinase (E.C.2.7.1.39)), such as a promoter truncation that reduces expression, aregrown under standard fungal growth conditions that are well known anddescribed in the art. A promoter truncation is made by deleting aportion of the promoter upstream of the transcription start site usingstandard molecular biology techniques that are well known and describedin the art (Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea fungalcells containing a mutant form of are grown under standard fungal growthconditions that are well known and described in the art. Magnaporthegrisea spores are harvested from cultures grown on complete agar mediumcontaining 4 mM L-threonine (Sigma-Aldrich Co.) after growth for 10-13days in the light at 25° C. using a moistened cotton swab. Theconcentration of spores is determined using a hemacytometer and sporesuspensions are prepared in a minimal growth medium to a concentrationof 2×10⁵ spores per ml. Approximately 4×10⁴ spores or cells areharvested and added to each well of 96-well plates to which growth mediais added in addition to an amount of test compound (at varyingconcentrations). The total volume in each well is 200 μl. Wells with notest compound present, and wells without cells are included as controls.The plates are incubated at 25° C. for seven days and optical densitymeasurements at 590 nm are taken daily. Wild type cells are screenedunder the same conditions. The effect of each compound on the mutant andwild-type fungal strains is measured against the growth control and thepercent of inhibition is calculated as the OD₅₉₀ (fungal strain plustest compound)/OD₅₉₀ (growth control)×100. The percent of growthinhibition as a result of a test compound on a fungal strain and that onthe wild type cells are compared. Compounds that show differentialgrowth inhibition between the mutant and the wild type are identified aspotential antifungal compounds. Similar protocols may be found in Kirschand DiDomenico ((1994) Biotechnology 26: 177-221).

Example 16 In vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Fungal THR4 and a Second Fungal Strain Containing aHeterologous THR4 Gene

[0172] Wild-type Magnaporthe grisea fungal cells and M. grisea fungalcells lacking a functional THR4 gene and containing a Thr4 gene fromSaccharomyces cerevisiae (Genbank: 6319901, 50% sequence identity) aregrown under standard fungal growth conditions that are well known anddescribed in the art. A M. grisea strain carrying a heterologous THR4gene is made as follows:

[0173] A M. grisea strain is made with a nonfunctional THR4 gene, suchas one containing a transposon insertion in the native gene (seeExamples 4 and 5).

[0174] A construct containing a heterologous THR4 gene is made bycloning the Thr4 gene from Saccharomyces cerevisiae into a fungalexpression vector containing a trpC promoter and terminator (e.g.pCB1003, Carroll et al. (1994) Fungal Gen News Lett 41: 22) usingstandard molecular biology techniques that are well known and describedin the art (Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual).

[0175] The said construct is used to transform the M. grisea strainlacking a functional THR4 gene (see Example 5). Transformants areselected on minimal agar medium lacking L-threonine. Only transformantscarrying a functional THR4 gene will grow.

[0176] Wild-type strains of Magnaporthe grisea and strains containing aheterologous form of THR4 are grown under standard fungal growthconditions that are well known and described in the art. Magnaporthegrisea spores are harvested from cultures grown on complete agar mediumafter growth for 10-13 days in the light at 25° C. using a moistenedcotton swab. The concentration of spores is determined using ahemacytometer and spore suspensions are prepared in a minimal growthmedium to a concentration of 2×10⁵ spores per ml. Approximately 4×10⁴spores or cells are harvested and added to each well of 96-well platesto which growth media is added in addition to an amount of test compound(at varying concentrations). The total volume in each well is 200 μl.Wells with no test compound present, and wells without cells areincluded as controls. The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. The effectof each compound on the wild-type and heterologous fungal strains ismeasured against the growth control and the percent of inhibition iscalculated as the OD₅₉₀ (fungal strain plus test compound)/OD₅₉₀ (growthcontrol)×100. The percent of growth inhibition as a result of a testcompound on the wild-type and heterologous fungal strains are compared.Compounds that show differential growth inhibition between the wild-typeand heterologous strains are identified as potential antifungalcompounds with specificity to the native or heterologous THR4 geneproducts. Similar protocols may be found in Kirsch and DiDomenico((1994) Biotechnology 26: 177-221).

Example 17 Pathway Specific in vivo Assay Screening Protocol

[0177]Magnaporthe grisea fungal cells are grown under standard fungalgrowth conditions that are well known and described in the art.Wild-type M. grisea spores are harvested from cultures grown on oatmealagar media after growth for 10-13 days in the light at 25° C. using amoistened cotton swab. The concentration of spores is determined using ahemocytometer and spore suspensions are prepared in a minimal growthmedium and a minimal growth medium containing 4 mM L-threonine(Sigma-Aldrich Co.) to a concentration of 2×10⁵ spores per ml. Theminimal growth media contains carbon, nitrogen, phosphate, and sulfatesources, and magnesium, calcium, and trace elements (for example, seeinoculating fluid in Example 7). Spore suspensions are added to eachwell of a 96-well microtiter plate (approximately 4×10⁴ spores/well).For each well containing a spore suspension in minimal media, anadditional well is present containing a spore suspension in minimalmedium containing 4 mM L-threonine. Test compounds are added to wellscontaining spores in minimal media and minimal media containingL-threonine. The total volume in each well is 200 μl. Both minimal mediaand L-threonine containing media wells with no test compound areprovided as controls. The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. A compoundis identified as a candidate for an antibiotic acting against theL-threonine biosynthetic pathway when the observed growth in the wellcontaining minimal media is less than the observed growth in the wellcontaining L-threonine as a result of the addition of the test compound.Similar protocols may be found in Kirsch and DiDomenico ((1994)Biotechnology 26: 177-221).

[0178] While the foregoing describes certain embodiments of theinvention, it will be understood by those skilled in the art thatvariations and modifications may be made and still fall within the scopeof the invention. The foregoing examples are intended to exemplifyvarious specific embodiments of the invention and do not limit its scopein any manner.

1 3 1 1650 DNA Magnaporthe grisea 1 atggagaacg gtgctgcaac caacggggcgtcggagaagt cgcactctcc ttcacagacc 60 tacctctcca caaggggaga cgattatgggctctcattcg agaccgtcgt cctcaaaggt 120 cttgcggctg acgggggtct tttcctgcccgaggaagtgc ccgcggcaac cgagtggcaa 180 agctggaaag acctgcccta caccgagcttgccgtcaagg ttctcagctt gtacatctcc 240 cccgccgagg tgccgacgga agacctcagggcgctcgtcg agcgcagcta ctcgaccttc 300 cgatccaagg aggttgtgcc gctggtgaagctggaggaca accttcacct gctggagcta 360 ttccacggcc ccagctactc gttcaaggactgcgcgctgc aattccttgg taacctcttc 420 gagtactttt tgactcgcaa gaacaagggaaaggagggca aagacaggca ccacctcact 480 gtggtcggcg caacaagtgg tgataccggttcggcggcca tctatggtct tcgcaacaag 540 aaggatgttt ccgtcttcat cctgcaccccaagggtcgtg taagccccat ccaggaggcc 600 cagatgacca cggtgctcga ccaaaatgttcacaaccttg ccgtgaccgg cacctttgac 660 gattgccaag atatcgtcaa ggccatgttcaacgacccag attcgaatgc gacactgaag 720 cttggtgctg tcaactcgat caactggtccaggatattgg cccagattgt ttactacttc 780 cactcgtact tttctctggc cagggcgtcaccagagacgt tcaaggtcgg cgacaaagtc 840 cgctttgtca cccccaccgg gaactttggtaacatcctgg ctggatactt tgcacaaaag 900 atgggcttgc ctgtcgacaa gttggtcgttgcgacaaatg agaacgacat tcttgacagg 960 ttttggaaga cgggccgcta cgaaaagaagcctgcaagcc ccgaggaagc cgcaggcggt 1020 ctgcctcaag atggcgtaaa ggctcacgaggagggctgca aggagaccct gagcccggcg 1080 atggacattt tggtgtcgag caactttgagcgaacactgt ggtttcttgc caaggagttc 1140 gctgctacgg ctggcctcaa tgacgagttcaacaagaagc aagccggcca ggaagttgtg 1200 gcatggtaca agtccctcaa ggctaccggaggcttcggtc cggtccaccc tgaaatcatg 1260 gacaatggcc gccaggtctt tgaaagcgagcgcgtgagcg acacccagac cctcgagatg 1320 atcgcggaga tgtacaaagc cacaaagtacgttctcgacc cgcactctgc cgtcggtgtt 1380 gcgggggcca agaggtcaat gtcgagggcctccaacgtcc cgcacatcgc gctgtccacg 1440 gcccacccag ccaagttctc tggcgccgttgagcttgcgc tcaaggacca gaaggagttc 1500 gactttacaa agcaggtcct gccagaggactttgttggac tagcagagaa ggaaaagagg 1560 gtgactgagg tggccgcgaa ctggcaggaagtgagggaga ttgtcaagaa gcaggtcgag 1620 gaagacttga aggctgaaag tagtgcataa1650 2 2082 DNA Magnaporthe grisea 2 tacgctgtca aataggcgat ggccgattacctattttgta ttgacaaaaa atgacaagac 60 cagctgtatc cactgatatc gataaggttttttattactg gccgatgtcg ggagacgcgg 120 ggcgaggtgg gcgaaattga ctaacactgattttgactga tgcgactgat gcgacagccg 180 cgcgacaaca cccaacacgc agacttgacagattctgcta ctacaaatcc tgcatattta 240 acagcgctgc aactcgacga tggagaacggtgctgcaacc aacggggcgt cggagaagtc 300 gcactctcct tcacagacct acctctccacaaggggagac gattatgggc tctcattcga 360 gaccgtcgtc ctcaaaggtc ttgcggctgacgggggtctt ttcctgcccg aggaagtgcc 420 cgcggcaacc gagtggcaaa gctggaaagacctgccctac accgagcttg ccgtcaaggt 480 tctcagcttg tacatctccc ccgccgaggtgccgacggaa gacctcaggg cgctcgtcga 540 gcgcagctac tcgaccttcc gatccaaggaggttgtgccg ctggtgaagc tggaggacaa 600 ccttcacctg ctggagctat tccacggccccagctactcg ttcaaggact gcgcgctgca 660 attccttggt aacctcttcg agtactttttgactcgcaag aacaagggaa aggagggcaa 720 agacaggcac cacctcactg tggtcggcgcaacaagtggt gataccggtt cggcggccat 780 ctatggtctt cgcaacaaga aggatgtttccgtcttcatc ctgcacccca agggtcgtgt 840 aagccccatc caggaggccc agatgaccacggtgctcgac caaaatgttc acaaccttgc 900 cgtgaccggc acctttgacg attgccaagatatcgtcaag gccatgttca acgacccaga 960 ttcgaatgcg acactgaagc ttggtgctgtcaactcgatc aactggtcca ggatattggc 1020 ccagattgtt tactacttcc actcgtacttttctctggcc agggcgtcac cagagacgtt 1080 caaggtcggc gacaaagtcc gctttgtcacccccaccggg aactttggta acatcctggc 1140 tggatacttt gcacaaaaga tgggcttgcctgtcgacaag ttggtcgttg cgacaaatga 1200 gaacgacatt cttgacaggt tttggaagacgggccgctac gaaaagaagc ctgcaagccc 1260 cgaggaagcc gcaggcggtc tgcctcaagatggcgtaaag gctcacgagg agggctgcaa 1320 ggagaccctg agcccggcga tggacattttggtgtcgagc aactttgagc gaacactgtg 1380 gtttcttgcc aaggagttcg ctgctacggctggcctcaat gacgagttca acaagaagca 1440 agccggccag gaagttgtgg catggtacaagtccctcaag gctaccggag gcttcggtcc 1500 ggtccaccct gaaatcatgg acaatggccgccaggtcttt gaaagcgagc gcgtgagcga 1560 cacccagacc ctcgagatga tcgcggagatgtacaaagcc acaaagtacg ttctcgaccc 1620 gcactctgcc gtcggtgttg cgggggccaagaggtcaatg tcgagggcct ccaacgtccc 1680 gcacatcgcg ctgtccacgg cccacccagccaagttctct ggcgccgttg agcttgcgct 1740 caaggaccag aaggagttcg actttacaaagcaggtcctg ccagaggact ttgttggact 1800 agcagagaag gaaaagaggg tgactgaggtggccgcgaac tggcaggaag tgagggagat 1860 tgtcaagaag caggtcgagg aagacttgaaggctgaaagt agtgcataat cacgagccgg 1920 agtgcagtag aaaatggtgt cgagatcagcatctagattt gctttcctag agatatgcaa 1980 acatttactt attctggacc ctgaatgcagccccaagggt gcactagatc ggataactgg 2040 aggtttagac gcggccgact tttccggaggtttttgaaag gg 2082 3 549 PRT Magnaporthe grisea 3 Met Glu Asn Gly AlaAla Thr Asn Gly Ala Ser Glu Lys Ser His Ser 1 5 10 15 Pro Ser Gln ThrTyr Leu Ser Thr Arg Gly Asp Asp Tyr Gly Leu Ser 20 25 30 Phe Glu Thr ValVal Leu Lys Gly Leu Ala Ala Asp Gly Gly Leu Phe 35 40 45 Leu Pro Glu GluVal Pro Ala Ala Thr Glu Trp Gln Ser Trp Lys Asp 50 55 60 Leu Pro Tyr ThrGlu Leu Ala Val Lys Val Leu Ser Leu Tyr Ile Ser 65 70 75 80 Pro Ala GluVal Pro Thr Glu Asp Leu Arg Ala Leu Val Glu Arg Ser 85 90 95 Tyr Ser ThrPhe Arg Ser Lys Glu Val Val Pro Leu Val Lys Leu Glu 100 105 110 Asp AsnLeu His Leu Leu Glu Leu Phe His Gly Pro Ser Tyr Ser Phe 115 120 125 LysAsp Cys Ala Leu Gln Phe Leu Gly Asn Leu Phe Glu Tyr Phe Leu 130 135 140Thr Arg Lys Asn Lys Gly Lys Glu Gly Lys Asp Arg His His Leu Thr 145 150155 160 Val Val Gly Ala Thr Ser Gly Asp Thr Gly Ser Ala Ala Ile Tyr Gly165 170 175 Leu Arg Asn Lys Lys Asp Val Ser Val Phe Ile Leu His Pro LysGly 180 185 190 Arg Val Ser Pro Ile Gln Glu Ala Gln Met Thr Thr Val LeuAsp Gln 195 200 205 Asn Val His Asn Leu Ala Val Thr Gly Thr Phe Asp AspCys Gln Asp 210 215 220 Ile Val Lys Ala Met Phe Asn Asp Pro Asp Ser AsnAla Thr Leu Lys 225 230 235 240 Leu Gly Ala Val Asn Ser Ile Asn Trp SerArg Ile Leu Ala Gln Ile 245 250 255 Val Tyr Tyr Phe His Ser Tyr Phe SerLeu Ala Arg Ala Ser Pro Glu 260 265 270 Thr Phe Lys Val Gly Asp Lys ValArg Phe Val Thr Pro Thr Gly Asn 275 280 285 Phe Gly Asn Ile Leu Ala GlyTyr Phe Ala Gln Lys Met Gly Leu Pro 290 295 300 Val Asp Lys Leu Val ValAla Thr Asn Glu Asn Asp Ile Leu Asp Arg 305 310 315 320 Phe Trp Lys ThrGly Arg Tyr Glu Lys Lys Pro Ala Ser Pro Glu Glu 325 330 335 Ala Ala GlyGly Leu Pro Gln Asp Gly Val Lys Ala His Glu Glu Gly 340 345 350 Cys LysGlu Thr Leu Ser Pro Ala Met Asp Ile Leu Val Ser Ser Asn 355 360 365 PheGlu Arg Thr Leu Trp Phe Leu Ala Lys Glu Phe Ala Ala Thr Ala 370 375 380Gly Leu Asn Asp Glu Phe Asn Lys Lys Gln Ala Gly Gln Glu Val Val 385 390395 400 Ala Trp Tyr Lys Ser Leu Lys Ala Thr Gly Gly Phe Gly Pro Val His405 410 415 Pro Glu Ile Met Asp Asn Gly Arg Gln Val Phe Glu Ser Glu ArgVal 420 425 430 Ser Asp Thr Gln Thr Leu Glu Met Ile Ala Glu Met Tyr LysAla Thr 435 440 445 Lys Tyr Val Leu Asp Pro His Ser Ala Val Gly Val AlaGly Ala Lys 450 455 460 Arg Ser Met Ser Arg Ala Ser Asn Val Pro His IleAla Leu Ser Thr 465 470 475 480 Ala His Pro Ala Lys Phe Ser Gly Ala ValGlu Leu Ala Leu Lys Asp 485 490 495 Gln Lys Glu Phe Asp Phe Thr Lys GlnVal Leu Pro Glu Asp Phe Val 500 505 510 Gly Leu Ala Glu Lys Glu Lys ArgVal Thr Glu Val Ala Ala Asn Trp 515 520 525 Gln Glu Val Arg Glu Ile ValLys Lys Gln Val Glu Glu Asp Leu Lys 530 535 540 Ala Glu Ser Ser Ala 545

What is claimed is:
 1. A method for identifying a test compound as acandidate for an antibiotic, comprising: a) contacting a Threoninesynthase polypeptide with said test compound; and b) detecting thepresence or absence of binding between a test compound and saidThreonine synthase polypeptide, wherein binding indicates that said testcompound is a candidate for an antibiotic.
 2. The method of claim 1,wherein said Threonine synthase polypeptide is a fungal Threoninesynthase polypeptide.
 3. The method of claim 1, wherein said Threoninesynthase polypeptide is a Magnaporthe Threonine synthase polypeptide. 4.The method of claim 1, wherein said Threonine synthase polypeptide isSEQ ID NO:
 3. 5. A method for determining whether the antibioticcandidate of claim 1 has antifungal activity, further comprisingcontacting a fungus or fungal cells with said antibiotic candidate anddetecting the decrease in growth, viability, or pathogenicity of saidfungus or fungal cells.
 6. A method for identifying a test compound as acandidate for an antibiotic, comprising: a) contacting a test compoundwith at least one polypeptide selected from the group consisting of: apolypeptide having at least ten consecutive amino acids of a fungalThreonine synthase, and a polypeptide having at least 50% sequenceidentity with a fungal Threonine synthase, and a polypeptide having atleast 10% of the activity of a fungal Threonine synthase; and b)detecting the presence and/or absence of binding between said testcompound and said polypeptide, wherein binding indicates that said testcompound is a candidate for an antibiotic.
 7. A method for determiningwhether the antibiotic candidate of claim 6 has antifungal activity,further comprising contacting a fungus or fungal cells with saidantibiotic candidate and detecting a decrease in growth, viability, orpathogenicity of said fungus or fungal cells.
 8. A method foridentifying a test compound as a candidate for an antibiotic,comprising: a) contacting O-phospho-L-homoserine and water with aThreonine synthase; b) contacting O-phospho-L-homoserine and water withThreonine synthase and a test compound; and c) determining the change inconcentration for at least one of the following: O-phospho-L-homoserine,L-threonine, orthophosphate, and water, wherein a change inconcentration for any of the above substances between steps (a) and (b)indicates that said test compound is a candidate for an antibiotic. 9.The method of claim 8, wherein said Threonine synthase is a fungalThreonine synthase.
 10. The method of claim 8, wherein said Threoninesynthase is a Magnaporthe Threonine synthase.
 11. The method of claim 8,wherein said Threonine synthase is SEQ ID NO:
 3. 12. A method fordetermining whether the antibiotic candidate of claim 8 has antifungalactivity, further comprising contacting a fungus or fungal cells withsaid antibiotic candidate and detecting a decrease in growth, viability,or pathogenicity of said fungus or fungal cells.
 13. A method foridentifying a test compound as a candidate for an antibiotic,comprising: a) contacting L-threonine and orthophosphate with aThreonine synthase; b) contacting L-threonine and orthophosphate with aThreonine synthase and a test compound; and c) determining the change inconcentration for at least one of the following: O-phospho-L-homoserine,L-threonine, orthophosphate, and water, wherein a change inconcentration for any of the above substances between steps (a) and (b)indicates that said test compound is a candidate for an antibiotic. 14.The method of claim 13, wherein said Threonine synthase is a fungalThreonine synthase.
 15. The method of claim 13, wherein said Threoninesynthase is a Magnaporthe Threonine synthase.
 16. The method of claim13, wherein said Threonine synthase is SEQ ID NO:
 3. 17. A method fordetermining whether the antibiotic candidate of claim 13 has antifungalactivity, further comprising contacting a fungus or fungal cells withsaid antibiotic candidate and detecting a decrease in growth, viability,or pathogenicity of said fungus or fungal cells.
 18. A method foridentifying a test compound as a candidate for an antibiotic,comprising: a) contacting O-phospho-L-homoserine and water with apolypeptide selected from the group consisting of: a polypeptide havingat least 50% sequence identity with Threonine synthase, and apolypeptide having at least 50% sequence identity with a Threoninesynthase and having at least 10% of the activity thereof, and apolypeptide comprising at least 100 consecutive amino acids of aThreonine synthase; b) contacting O-phospho-L-homoserine and water withsaid polypeptide and a test compound; and c) determining the change inconcentration for at least one of the following: O-phospho-L-homoserine,L-threonine, orthophosphate, and water, wherein a change inconcentration for any of the above substances between steps (a) and (b)indicates that said test compound is a candidate for an antibiotic. 19.A method for identifying a test compound as a candidate for anantibiotic, comprising: a) contacting L-threonine and orthophosphatewith a polypeptide selected from the group consisting of: a polypeptidehaving at least 50% sequence identity with a Threonine synthase, and apolypeptide having at least 50% sequence identity with a Threoninesynthase and at least 10% of the activity thereof, and a polypeptidecomprising at least 100 consecutive amino acids of a Threonine synthase;b) contacting L-threonine and orthophosphate, with said polypeptide anda test compound; and c) determining the change in concentration for atleast one of the following: O-phospho-L-homoserine, L-threonine,orthophosphate, and water, wherein a change in concentration for any ofthe above substances between steps (a) and (b) indicates that said testcompound is a candidate for an antibiotic.
 20. A method for identifyinga test compound as a candidate for an antibiotic, comprising: a)measuring the expression of a Threonine synthase in a cell, cells,tissue, or an organism in the absence of a test compound; b) contactingsaid cell, cells, tissue, or organism with said test compound andmeasuring the expression of said Threonine synthase in said cell, cells,tissue, or organism; and c) comparing the expression of Threoninesynthase in steps (a) and (b), wherein a lower expression in thepresence of said test compound indicates that said test compound is acandidate for an antibiotic.
 21. The method of claim 20, wherein saidcell, cells, tissue, or organism is, or is derived from a fungus. 22.The method of claim 20, wherein said cell, cells, tissue, or organismis, or is derived from a Magnaporthe fungus or fungal cell.
 23. Themethod of claim 20, wherein said Threonine synthase is SEQ ID NO:
 3. 24.The method of claim 20, wherein the expression of Threonine synthase ismeasured by detecting THR4 mRNA.
 25. The method of claim 20, wherein theexpression of Threonine synthase is measured by detecting Threoninesynthase polypeptide.
 26. A method for identifying a test compound as acandidate for an antibiotic, comprising: a) providing cells having oneform of a Threonine synthase gene, and providing comparison cells havinga different form of a Threonine synthase gene; and b) contacting saidcells and said comparison cells with a test compound and determining thegrowth of said cells and comparison cells in the presence of the testcompound, wherein a difference in growth between said cells and saidcomparison cells in the presence of said compound indicates that saidcompound is a candidate for an antibiotic.
 27. The method of claim 26wherein the cells and the comparison cells are fungal cells.
 28. Themethod of claim 26 wherein the cells and the comparison cells areMagnaporthe cells.
 29. The method of claim 26 wherein said form and saiddifferent form of the Threonine synthase are fungal Threonine synthases.30. The method of claim 26, wherein at least one of the forms is aMagnaporthe Threonine synthase.
 31. The method of claim 26, wherein saidform and said different form of the Threonine synthase are non-fungalThreonine synthases.
 32. The method of claim 26, wherein one form of theThreonine synthase is a fungal Threonine synthase, and the differentform is a non-fungal Threonine synthase.
 33. A method for identifying atest compound as a candidate for an antibiotic, comprising: a) providingcells having one form of a gene in the L-threonine biochemical and/orgenetic pathway and providing comparison cells having a different formof said gene; b) contacting said cells and said comparison cells with asaid test compound; and c) determining the growth of said cells and saidcomparison cells in the presence of said test compound, wherein adifference in growth between said cells and said comparison cells in thepresence of said test compound indicates that said test compound is acandidate for an antibiotic.
 34. The method of claim 33, wherein thecells and the comparison cells are fungal cells.
 35. The method of claim33, wherein the cells and the comparison cells are Magnaporthe cells.36. The method of claim 33, wherein said form and said different form ofthe L-threonine biosynthesis gene are fungal L-threonine biosynthesisgenes.
 37. The method of claim 33, wherein at least one of the forms isa Magnaporthe L-threonine biosynthesis gene.
 38. The method of claim 33,wherein said form and said different form of the L-threoninebiosynthesis genes are non-fungal L-threonine biosynthesis genes. 39.The method of claim 33, wherein one form of the L-threonine biosynthesisgene is a fungal L-threonine biosynthesis gene, and the different formis a non-fungal L-threonine biosynthesis gene.
 40. A method fordetermining whether the antibiotic candidate of claim 33 has antifungalactivity, further comprising contacting a fungus or fungal cells withsaid antibiotic candidate and detecting a decrease in growth, viability,or pathogenicity of said fungus or fungal cells, wherein a decrease ingrowth, viability, or pathogenicity of said fungus or fungal cellsindicates that the antibiotic candidate has antifungal activity.
 41. Amethod for identifying a test compound as a candidate for an antibiotic,comprising: (a) providing paired growth media comprising a first mediumand a second medium, wherein said second medium contains a higher levelof L-threonine than said first medium; (b) contacting an organism with atest compound; (c) inoculating said first and said second media withsaid organism; and (d) determining the growth of said organism, whereina difference in growth of the organism between said first and saidsecond media indicates that said test compound is a candidate for anantibiotic.
 42. The method of claim 41, wherein said organism is afungus.
 43. The method of claim 41, wherein said organism isMagnaporthe.
 44. An isolated nucleic acid comprising a nucleotidesequence that encodes a polypeptide of SEQ ID NO:
 3. 45. The nucleicacid of claim 44 comprising the nucleotide sequence of SEQ ID NO:
 1. 46.An expression cassette comprising the nucleic acid of claim
 45. 47. Theisolated nucleic acid of claim 44 comprising a nucleotide sequence withat least 50 to at least 95% sequence identity to SEQ ID NO:
 1. 48. Apolypeptide consisting essentially of the amino acid sequence of SEQ IDNO:
 3. 49. A polypeptide comprising the amino acid sequence of SEQ IDNO: 3.